Listeria monocytogenes is a gram-positive, opportunistic, intracellular bacterial pathogen that causes food borne illness. Given its well-characterized infection cycle and genetic amenability, L. monocytogenes provides a powerful tool to interrogate the fundamental aspects of intracellular bacterial pathogenesis and the host immune response to invasion by intracellular pathogens. It has previously been demonstrated that L. monocytogenes that enter into the host cell cytosol secrete a signaling nucleotide, c-di-AMP, resulting in stimulation of host IFN- production. Our preliminary studies have uncovered important roles for c-di-AMP signaling in the ability of the bacterium to grow in broth and within the host. Furthermore, evidence suggests c-di-AMP may have functional consequences beyond IFN- induction by the infected host cell. Using proteomics and genetic studies we have identified novel bacterial and host pathways that may mediate the numerous phenotypes associated with this unique bacterial signaling molecule. In Aim I, we propose to explore the role of a bacterial small RNA regulated by c-di-AMP and required for Listeria virulence. In Aim II, we will characterize the role of three c-di-AMP binding proteins in mediating signal transduction. In the final aim, we will characterize the effects of c-di-AMP binding to a novel host protein and elucidate the effects of these interactions on the outcome of infection. Together these studies will define the molecular mechanisms of c-di-AMP signaling that mediate host pathogen interactions during infection by this intracellular bacterium.